Laboratory Diagnosis of Covid 19 Infection
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Introduction
SARS CoV 2 Virus
Covid 19 disease
Diagnostic analyte dynamics
Covid 19 case definition
Who to test (ICRM Guidelines)
Preanalytic: PPE Donning
Which sample to collect
Recovery of SARS CoV 2 from different samples
How long SARS CoV 2 survive on common surfaces
Doffing PPE
FDA allows self collection of nasal swab
Analytic: RT PCR
Pathkind Labs Dummy Report
Serology
ICMR Guidance on Rapid antibody kits
Covid 19 Ag GICA Rapid
Disposal of waste
Infection Control Precautions
Personal Protection Steps
Pathkind Labs Covid 19 Clinical Information Form
NABL Accreditation
ICRM List of approved Private Labs in Haryana
Frequent Q & A
Mask Protection Efficency
Rational Use of PPE for Covid 19 (WHO)
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8 – 10
11 – 13
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Introduction:
WHO declared an outbreak of febrile respiratory illness of unknown etiology in December
2019 from Wuhan, Hubei province of China. Since its emergence, the disease rapidly spread
to neighboring provinces of China as well as to 182 other countries. Infection is spread
through droplets of an infected patient generated by coughing and sneezing or through
prolonged contact with infected patients.
Coronavirus disease 2019 (COVID-19) is a potentially severe acute respiratory infection
caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus was
identified as the cause of an outbreak of pneumonia of unknown cause in Wuhan City,
Hubei Province, China, in December 2019. The clinical presentation is that of a respiratory
infection with a symptom severity ranging from a mild common cold-like illness, to a severe
viral pneumonia leading to acute respiratory distress syndrome that is potentially fatal.
The International Committee on Taxonomy of Viruses has confirmed SARS-CoV-2 as the
name of the virus owing to the virus's genetic similarity to the SARS-CoV virus, but taking
into account that there may be differences in disease spectrum and transmission. The World
Health Organization has confirmed COVID-19 (a shortened version of coronavirus disease
2019) as the name of the disease that SARS-CoV-2 infection causes. Prior to this, the virus
and/or disease was known by various names including novel coronavirus (2019-nCoV),
2019-nCoV, or variations on this.
Virus: SARS CoV 2
Several coronaviruses can infect humans, the globally endemic human coronaviruses HCoV229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43 that tend to cause mild respiratory disease,
and the zoonotic Middle East respiratory syndrome coronavirus (MERS-CoV) and severe
acute respiratory syndrome coronavirus (SARS-CoV) that have a higher case fatality rate. In
December 2019, a cluster of patients with a novel coronavirus was identified in Wuhan,
China . Initially tentatively named 2019 novel coronavirus (2019-nCoV), the virus has now
been named SARS-CoV-2 by the International Committee of Taxonomy of Viruses (ICTV) .
This virus can cause the disease named coronavirus disease 2019 (COVID-19).
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Disease:
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Diagnostic Analyte Dynamics:
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COVID-19 Case Definitions
Suspect Case:
A patient with acute respiratory illness (fever and at least one sign/ symptom of respiratory
disease (e.g., cough, shortness of breath) AND a history of travel to of residence in a
country/area or territory reporting local transmission (See NCDC website for updated list) of
COVID-19 disease during the 14 days prior to symptom onset;
OR
A patient / Health care worker with any acute respiratory illness AND having been in contact
with a confirmed COVID-19 case in the last 14 days prior to onset of symptoms;
OR
A patient with severe acute respiratory infection (fever and at least one sign/symptom of
respiratory disease (e.g., cough, shortness breath) AND requiring hospitalization AND with
no other etiology that fully explains the clinical presentation;
OR
A case for whom testing for COVID-19 is inconclusive
Laboratory Confirmed case: A person with laboratory confirmation of COVID-19 infection,
irrespective of clinical signs and symptoms.
Who to test:
Current testing strategy: (ICMR Advisory ver 3 wef 20 March 2020)
i. All asymptomatic individuals who have undertaken international travel in the last 14 days:
- They should stay in home quarantine for 14 days.
- They should be tested only if they become symptomatic (fever, cough, difficulty in
breathing)
- All family members living with a confirmed case should be home quarantined
ii. All symptomatic contacts of laboratory confirmed cases.
iii. All symptomatic health care workers.
iv. All hospitalized patients with Severe Acute Respiratory Illness (fever AND cough and/or
shortness of breath).
v. Asymptomatic direct and high-risk contacts of a confirmed case should be tested once
between day 5 and day 14 of coming in his/her contact.
- Direct and high-risk contact include those who live in the same household with a
confirmed case and healthcare workers who examined a confirmed case without adequate
protection as per WHO recommendations
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Preanalytic:
PPE: Donning
What sample to collect
Upper respiratory tract
Oropharyngeal swab (OP swab)
Use only synthetic fiber swabs with plastic shafts. Do not use calcium alginate swabs or
swabs with wooden shafts, as they may contain substances that inactivate some viruses
and inhibit PCR testing. Place swabs immediately into sterile tubes containing 2-3 ml of
viral transport media. Refrigerate specimen at 2-8°C and ship to Pathkind Gurgaon Lab
with ice pack.
Oropharyngeal swab (e.g., throat swab): Swab the posterior pharynx, avoiding the
tongue.
Insert a thin flexible swab through mouth over the tongue and turn the swab upwards
behind the soft palate to reach the nasopharynx.
Leave the swab in place for a few seconds.
Slowly remove swab and put the swab with tip downwards into vial containing VTM,
breaking the extra portion of the swab stick.
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Nasal aspirate
Collect 2-3 mL into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry
container. Refrigerate specimen at 2-8°C and ship to Pathkind Gurgaon Lab with ice
pack. It maybe best to take one throat and one nasal swab and put both into one VTM.
BD Universal Viral Transport Medium (VTM)
Recovery of SARS CoV 2 from different samples
(JAMA online March 11, 2020)
Specimen
Bronchalveolar
lavage (BAL)
Brush Biopsy
Sputum
Nasal swab
Pharyngeal swab
Faeces
Blood
Urine
Number tested
15
Number positive
14
Percentage positive
93
13
104
8
398
153
307
72
6
75
5
126
44
3
0
46
72
63
32
29
1
0
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Doffing PPE:
FDA allows self collection of Nasal Swab:
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Specimen Collection
FDA believes that a nasopharyngeal specimen is the preferred choice for
swab-based SARS-CoV-2 testing.
If a nasopharyngeal specimen is not available, then any of the following are
acceptable:



oropharyngeal specimen collected by a healthcare professional
(HCP);
mid-turbinate specimen by onsite self-collection or HCP (using a
flocked tapered swab); or
anterior nares specimen by onsite self-collection or HCP (using a
round foam swab).
Multiple specimens may be taken with a single swab. If a separate swab is
used for collecting specimens from two different locations in the same
patient, both swabs may be placed in the same vial in order to conserve
collection and assay supplies. At this time, anterior nares and midturbinate specimen collection are only appropriate for symptomatic
patients and both nares should be swabbed. There is currently not enough
information to recommend nasal or mid-turbinate testing for
asymptomatic persons.
Other swab specimens (i.e., tongue swabs) may have decreased sensitivity,
so caution should be exercised when interpreting negative results.
More data are necessary on the validity of buccal swabs or saliva specimens
alone.
For patients with productive cough, a sputum sample is an acceptable lower
respiratory specimen.
Due to concerns with specimen stability, transport, and appropriate
collection materials, self-collection at home or at sites other than
designated collection sites staffed by HCPs is currently not recommended.
FDA believes that sample collection with a flocked swab, when available, is
preferred. Collection should be conducted with a sterile swab. If the
applicator handle requires additional trimming, the trimming should be
performed with a sterile pair of scissors to prevent contamination of the
sample. Swab recommendations are based on limited available evidence,
and expert opinion suggests further research is needed in this area.
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Please be aware that the CDC does not recommend use of calcium alginate
swabs or swabs with wooden shafts, as they may contain substances that
inactivate some viruses and inhibit PCR testing.
To avoid specimens being wasted, if a lab is presented with a specimen that
was collected or identified in a sub-optimal manner, e.g. with a swab for
which there is less evidence of effectiveness, FDA believes that it would still
be appropriate for the lab to accept the specimen for analysis and note the
circumstances on the report. These specimens may have decreased
sensitivity, so caution should be exercised when interpreting negative
results.
Transport Media
VTM/UTM remains the preferred transport media.
In the absence of VTM/UTM, alternative transport media can be used to
collect and transport patient samples for molecular RT-PCR SARS-CoV-2
assays. These recommendations apply to swab-based specimen collection
by healthcare providers (HCP), and to anterior nares (nasal) and midturbinate specimen collection onsite by self-collection. The best available
evidence indicates that these transport media will stabilize the SARS-CoV-2
RNA without meaningful degradation.
Labs can create their own viral transport media. Refer to CDC's SOP#:
DSR-052-01: Preparation of Viral Transport Media. Specimens can be
stored for up to 72 hours at 4℃, or frozen for longer storage.
Liquid Amies media may be used for viral transport when universal
transport media is not available. Specimens can be stored in liquid Amies
media for up to 72 hours at 4℃, or frozen for longer storage. All of the
products listed below include a nasopharyngeal (NP) flocked swab unless
noted otherwise.
If the above are not available, FDA recommends use of a dry swab in saline
to collect and transport samples for molecular RT-PCR SARS-CoV-2 assays.
FDA believes that for saline, a sterile glass or plastic vial containing
between 1mL and 3mL of phosphate buffered saline is appropriate.
Specimens can be stored up to 72 hours at 4℃, or frozen for longer storage.
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Analytic:
RT PCR
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Pathkind Lab’s Dummy Report:
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Serology: Not Yet Recommended by ICMR
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ICMR Guidance on Rapid antibody kits for COVID-19
Not recommended for diagnosis of COVID-19 infection
Can be done on blood/serum/plasma samples
Test result is available within 30 minutes
Test comes positive after 7-10 days of infection
The test remains positive for several weeks after infection
Positive test indicates exposure to SARS-CoV-2
Negative test does not rule out COVID-19 infection
ICMR has approved a number of Antibody Rapid tests, but these tests are not
recommended for diagnosis of COVID-19 infection. Antigen detection may be useful
Covid 19 Ag GICA Rapid: Not Yet Available in India
Two Biotechnology companies in China and South Korea have constructed Antigen
detection tests using monoclonal antibodies constructed using phage display technology.
Covid 19 Ag GICA Rapid
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Disposal of waste:
Keep separate color coded bins/ bags / containers in collection area and maintain proper
segregation of waste as per BMWM Rules, 2016 as amended and CPCB guidelines for
implementation of BMW Management Rules.
As precaution double layered bags (using 2 bags) should be used for collection of waste
from COVID – 19 isolation wards so as to ensure adequate strength and no-leaks;
Collection and store biomedical waste separately prior to handing over the same
CBWTF.
Use a dedicated collection bin labelled as “COVID – 19” to store COVID – 19 waste. In
addition to mandatory labelling, bags/ containers used for collecting biomedical waste
from COVID – 19 wards, should be labelled as “COVID – 19 Waste”. This marking would
enable CBWTFs to identify the waste easily for priority treatment and disposal
immediately upon the receipt.
General waste not having contamination should be disposed as solid waste as per BMW
Rules 2016;
Maintain separated record of waste generated from COVID-19 isolation wards.
Use dedicated trolleys and collection bin in collection area. A label “COVID-19 Waste” to
be pasted on these items also.
The (inner and outer) surface of container/ bins/ trolleys used for storage of COVID – 19
waste should be disinfected with 1% sodium hypochlorite solution. Finally waste must be
autoclaved before being handed over to the authorized vendor for final disposal.
Infection Prevention:
IPC strategies to prevent or limit infection transmission in health-care settings include
the following:
1. Early recognition and source control
2. Application of Standard Precautions for all patients
3. Implementation of empiric additional precautions (droplet and contact and
whenever applicable airborne precautions) for suspected cases
4. Administrative controls
5. Environmental and engineering controls
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Personal protection steps:
Johns Hopkins University has sent this detailed note on avoiding the contagion:
* The virus is not a living organism, but a protein molecule (DNA) covered by a
protective layer of lipid (fat), which, when absorbed by the cells of the ocular, nasal
or buccal mucosa, changes their genetic code. (mutation) and convert them into
aggressor and multiplier cells.
* Since the virus is not a living organism but a protein molecule, it is not killed, but
decays on its own. The disintegration time depends on the temperature, humidity
and type of material where it lies.
* The virus is very fragile; the only thing that protects it is a thin outer layer of fat.
That is why any soap or detergent is the best remedy, because the foam CUTS the
FAT (that is why you have to rub so much: for 20 seconds or more, to make a lot of
foam). By dissolving the fat layer, the protein molecule disperses and breaks down
on its own.
* HEAT melts fat; this is why it is so good to use water above 25 degrees Celsius for
washing hands, clothes and everything. In addition, hot water makes more foam and
that makes it even more useful.
* Alcohol or any mixture with alcohol over 65% DISSOLVES ANY FAT, especially
the external lipid layer of the virus.
* Any mix with 1 part bleach and 5 parts water directly dissolves the protein, breaks it
down from the inside.
* Oxygenated water helps long after soap, alcohol and chlorine, because peroxide
dissolves the virus protein, but you have to use it pure and it hurts your skin.
* NO BACTERICIDE SERVES. The virus is not a living organism like bacteria; they
cannot kill what is not alive with anthobiotics, but quickly disintegrate its structure
with everything said.
* NEVER shake used or unused clothing, sheets or cloth. While it is glued to a
porous surface, it is very inert and disintegrates only between 3 hours (fabric and
porous), 4 hours (copper, because it is naturally antiseptic; and wood, because it
removes all the moisture and does not let it peel off and disintegrates). ), 24 hours
(cardboard), 42 hours (metal) and 72 hours (plastic). But if you shake it or use a
feather duster, the virus molecules float in the air for up to 3 hours, and can lodge in
your nose.
* The virus molecules remain very stable in external cold, or artificial as air
conditioners in houses and cars. They also need moisture to stay stable, and
especially darkness. Therefore, dehumidified, dry, warm and bright environments will
degrade it faster.
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* UV LIGHT on any object that may contain it breaks down the virus protein. For
example, to disinfect and reuse a mask is perfect. Be careful, it also breaks down
collagen (which is protein) in the skin, eventually causing wrinkles and skin cancer.
* The virus CANNOT go through healthy skin.
* Vinegar is NOT useful because it does not break down the protective layer of fat.
* NO SPIRITS, NOR VODKA, serve. The strongest vodka is 40% alcohol, and you
need 65%.
* LISTERINE IF IT SERVES! It is 65% alcohol.
* The more confined the space, the more concentration of the virus there can be.
The more open or naturally ventilated, the less.
* This is super said, but you have to wash your hands before and after touching
mucosa, food, locks, knobs, switches, remote control, cell phone, watches,
computers, desks, TV, etc. And when using the bathroom.
* You have to HUMIDIFY HANDS DRY from so much washing them, because the
molecules can hide in the micro cracks. The thicker the moisturizer, the better.
* Also keep your NAILS SHORT so that the virus does not hide there.
This is a cut & Paste version of various relevant information available from reliable sources on Covid 19
disease and SARS CoV 2 virus. The information is evolving and may change over time.
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COVID-19 CLINICAL INFORMATION FORM
AFFIX
BARCODE
(ICMR NO: __________)
Patient Details
Name of patient:
Age:
Gender:
Date of Birth: ___/___/_________
Permanent Address:
Current Address: Same as above
Nationality:
Mobile / Phone:
Email:
Date of sample collection:
Ref. Hospital / Doctor name & contact details:
Exposure History
Is the patient quarantined? Y/N
International Travel History: Y/N
If yes, Travel place: _____________ & Stay / travel duration with date from: ___/___/__________ to ___/___/__________
Health care worker working in hospital/ lab involved in managing patients: Y/N
Hospitalization date ___/___/__________ Discharge date ___/___/__________
Status of clinical symptoms - Symptomatic / Asymptotic
If Symptomatic date of onset of symptom:_____________
Symptoms
Please tick
whichever is
applicable
Symptoms
Fever ( <7 days )
Vomiting
Fever ( >7 days )
Muscle pain
Cough
Abdominal pain
Difficulty in Breathing
Headache
Nausea
Diarrhoea
Please tick
whichever is
applicable
Any other symptom (please mention with date onset):_____________
If any other tests done (please provide details )
Specimen Information
Type of sample:
Nasal and Throat swab
Any other (please mention):
Valid Govt. ID attached showing address proof : ______________________________
(Specify: Aadhar/Voter ID/Driving License /Passport
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Question & Answers:
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